Fabrication of MERS-nanovesicle biosensor composed of multi-functional DNA aptamer/graphene-MoS2 nanocomposite based on electrochemical and surface-enhanced Raman spectroscopy

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is one of the most harmful viruses for humans in nowadays. To prevent the spread of MERS-CoV, a valid detection method is highly needed. For the first time, a MERS-nanovesicle (NV) biosensor composed of multi-functional DNA aptamer and graphene oxide encapsulated molybdenum disulfide (GO-MoS₂) hybrid nanocomposite was fabricated based on electrochemical (EC) and surface-enhanced Raman spectroscopy (SERS) techniques. The MERS-NV aptamer was designed for specifically binding to the spike protein on MERS-NVs and it is prepared using the systematic evolution of ligands by exponential enrichment (SELEX) technique. For constructing a multi-functional MERS aptamer (MF-aptamer), the prepared aptamer was connected to the DNA 3-way junction (3WJ) structure. DNA 3WJ has the three arms that can connect the three individual functional groups including MERS aptamer (bioprobe), methylene blue (signal reporter) and thiol group (linker) Then, GO-MoS₂ hybrid nanocomposite was prepared for the substrate of EC/SERS-based MERS-NV biosensor construction. Then, the assembled multifunctional (MF) DNA aptamer was immobilized on GO-MoS₂. The proposed biosensor can detect MERS-NVs not only in a phosphate-buffered saline (PBS) solution (SERS LOD: 0.176 pg/ml, EIS LOD: 0.405 pg/ml) but also in diluted 10% saliva (SERS LOD: 0.525 pg/ml, EIS LOD: 0.645 pg/ml).

Keywords: Aptamer; MERS-NV; MERS-NV biosensor; SELEX; Surface-enhanced Raman spectroscopy.

Fig. 1

Schematic image of the fabricated MERS-NV detection biosensor based on EC/SERS method.

Fig. 2

(a) Native-TBE PAGE gel result: SELEX product binding affinity test with S1 protein and control protein, (b) Expected 2D structure of MERS S1 aptamer, (c) Native-TBE PAGE gel result: identified MERS aptamer binding affinity test with spike protein and control protein, (d) Fluorescence binding assay results between FAM-tagged MERS aptamer and S1 protein, (e) Expected 2D structure of multi-functional MERS aptamer, (f) Native-TBM PAGE gel result: MF-MERS aptamer assembly, (g) Native-TBM PAGE gel result: MF-MERS aptamer binding affinity test with the MERS-NV and control proteins (myoglobin and HA protein).

Fig. 3

(a) Schematic diagram of GO-MoS₂, MF MERS aptamer on GO-MoS₂, MERS-NV/MF MERS aptamer on GO-MoS₂ layers, respectively. AFM results of (b) surface morphology of GO-MoS₂, (c) surface morphology of MF MERS aptamer on GO-MoS₂ (d) Surface morphology of MERS-NV on MF MERS aptamer, (e) Surface roughness analysis results. (f) SERS spectra of the GO-MoS₂ nanocomposite (black line), MF MERS aptamer on GO-MoS₂ (red line), MERS-NV reacted with MF MERS aptamer on GO-MoS₂ (blue line), respectively. (g) Nyquist plot of the GO-MoS₂ nanocomposite (red dot line), MF MERS aptamer on GO-MoS₂ (blue dot line), MERS-NV reacted with MF MERS aptamer on GO-MoS₂ (green dot line), respectively.

Fig. 4

(a) SERS spectra of MERS-NV detection in PBS buffer through the fabricated MF MERS aptamer/GO-MoS₂ (1 pg/ml to 100 ng/ml), (b) Linear curve of the different concentration of MERS-NV range from 1 pg/ml to 100 ng/ml with correlation coefficient (R²) of 0.992, (c) SERS spectra of MERS-NV detection in 10% saliva through the fabricated MF MERS aptamer/GO-MoS₂ (1 pg/ml to 100 ng/ml), (d) Linear curve of the different concentration of MERS-NV range from 1 pg/ml to 100 ng/ml with correlation coefficient (R²) of 0.991, (e) Selectivity test of MF MERS aptamer/GO-MoS₂-based SERS biosensor with the various controls. (f) Plot of SERS intensity versus various proteins. All selectivity tests were performed in 10% saliva. Error bar represents the relative standard deviation of 10 independent experiments.

Fig. 5

(a) EIS results of MERS-NV detection in PBS buffer (1 pg/ml to 100 ng/ml), (b) Linear curve of the different concentration of MERS-NV range from 1 pg/ml to 100 ng/ml with correlation coefficient (R²) of 0.989, (c) EIS results of MERS-NV detection in 10% saliva (1 pg/ml to 100 ng/ml), (d) Linear curve of the different concentration of MERS-NV range from 1 pg/ml to 100 ng/ml with correlation coefficient (R²) of 0.988. (e) Selectivity test with the various targets through EIS. (f) ΔR_ct values based on selectivity test with other protein reaction targets; H5N1 HA protein, H1N1 HA protein, hemoglobin, amylase, immunoglobulin G and MERS-NV. Error bar represents the relative standard deviation of 10 independent experiments.